The small intestinal epithelium renews every 3-5 days. To maintain this high rate of cell loss, new cells are continuously generated from a population of multi-potent intestinal stem cells (ISCs). A more complete understanding of the molecular mechanisms that regulate ISC function has enormous implications for the study of gut injury repair, normal homeostasis and gastrointestinal cancer. Although the precise identity and location of ISCs remains under intense debate, recent studies using elegant reporter mouse models have suggested that two distinct ISC populations exist, one quiescent population, located at the +4 position relative to the crypt bottom just above the Paneth cells. The other stem cell population is characterized by rapidly cycling crypt base columnar cells (CBCs) interspersed between the Paneth cells. These +4 cells have been implicated in a rescue stem cell response following radiation injury (IR). We have previously reported that the putative ISC marker Dclk1 (formerly known as DCAMKL-1) is predominantly expressed in the +4 position in the crypt is relatively quiescent under basal conditions and is mostly distinct from the proliferative stem cells (CBCs). Gastrointestinal stem cells are increasingly being recognized as potential cells of origin in colon cancer. It has been demonstrated that DCLK1 is upregulated in human colorectal cancers (CRC) and can be used as a prognostic marker. Interestingly siRNA-mediated knockdown of DCLK1 results in colorectal tumor xenograft growth arrest, inhibition of c-Myc, KRAS via let-7a miRNA, Notch-1 via miR-144 and epithelial-mesenchymal transition (EMT) associated factors via miR-200a. The central hypothesis of this VA Merit proposal is that Dclk1 plays a key role in regulating the rescue stem cell population in response to severe genotoxic and potentially mutagenic injury, and these rescue/reserve cells are potential cells of origin that can contribute to gastrointestinal neoplasia. To test this hypothesis, we propose 3 specific aims: Aim 1: To determine whether Dclk1 regulates small intestinal and colonic regenerative responses after injury via genes related to Wnt and Notch signaling, EMT and epithelial barrier function. Dclk1 knockout (Villin-Cre; Dclk1flox/flox) and wild type mice will be subjected to: (a) whole body IR (genotoxic injury) to evaluate the effects of deletion of +4 rescue stem cell population in the crypt regenerative response; (b) DSS- induced colitis (cytotoxic injury) and (c) Azoxymethane (AOM)+DSS (mutagenic insult) to evaluate the effects of epithelial Dclk1 knockout in colonic crypt regeneration and tumorigenesis. Aim 2: To determine whether Dclk1+ cell is the origin of the regenerative crypt and cell of origin of cancer after genotoxic/cytotoxic injury and mutagenic insult respectively. Using a reporter mouse model (Dclk1-Cre-ERTM;Rosa26GFP), where GFP is expressed in Dclk1+ cells and their progeny, lineage tracing studies will be performed. We will examine the effects of genotoxic, cytotoxic and mutagenic injuries on this mouse model to determine whether the Dclk1+ cell is the cell of origin of regenerative crypts and cancer. Aim 3: To determine whether DCLK1 can be used as an early diagnostic marker for colorectal neoplasia (i.e. adenoma) detection. Here, we will determine whether DCLK1 can be detected in the circulation of patients with colorectal neoplasia and correlate with tissue immunostaining. We will also determine CSC properties of DCLK1+ cells isolated from human CRC blood samples. Furthermore, DCLK1 potentially can be used as a tissue and serum diagnostic marker of early colorectal neoplasia. CRC continues to be a major cause of morbidity and mortality in the veteran population and there is emerging evidence that cancer stem cells are the key initiators of colon cancer. These studies have the major potential to fill a major unmet medical need.